Pyrogens are designated as inflammatory acting substances that can induce fever. A differentiation is made in this case between bacterial pathogens, viral pathogens, pyrogens of fungi and pyrogens of a non-biological origin, such as e.g., metal compounds in elastomers, rubber abrasion or microscopic plastic particles from medical technology products or pharmaceuticals. Among the bacterial pyrogens a differentiation is made between endotoxins from the membrane of gram-negative bacteria, such as e.g., lipopolysaccharides (LPS), and components of gram-positive bacteria, such as e.g., lipoteichoic acids.
Pyrogens that have invaded the body stimulate immune cells that are capable of phagocytosis to synthesize proinflammatory cytokines, in particular interleukins (primarily IL-1 and IL-6) and tumor necrosis factor-a (TNF-a), which then as the “actual pyrogens” influence the temperature center of the body in such a way that increased heat production and reduced heat dissipation occur. The immune system is thus stimulated and normally the invading microorganism is eliminated. However, pathogenic microbes and products thereof, i.e., pyrogens, can in some circumstances invade the bloodstream from a source of infection and thus systemically activate an inflammatory cascade, which produces a systemic inflammatory response that is no longer controlled. The clinical complex of symptoms associated with this inflammatory response is designated as sepsis or colloquially as “blood poisoning”. Since 1992, four separate degrees of severity have been differentiated in this connection: systemic inflammatory response syndrome (SIRS), sepsis, severe sepsis and septic shock. Sepsis as such is defined as SIRS with a confirmed infection.
In the course of sepsis, frequently a life-threatening impairment of vital functions occurs and the failure of one or more organs (multi-organ failure). Intensive care can bridge critical phases by temporarily replacing or supporting organ functions (ventilation, renal replacement therapy, cardiovascular therapy, coagulation therapy). Despite this, sepsis must be classified as a very severe illness with an extremely serious prognosis: 30-50% of those afflicted die despite maximum treatment. The earliest possible start of treatment is crucial for survival. In Germany 150,000 people fall ill with sepsis every year; 56,300 of them die.
The prognosis is particularly unfavorable when treatment is started late, sources of infection cannot be localized, or pathogens cannot be identified. Therefore, rapid and reliable identification of the pathogens or the pathogen spectra is of great significance for initiating a targeted treatment of the infection in clinical diagnostics in hospitals.
Most of the time, pathogens that cause sepsis are bacteria, predominantly gram-negative bacteria like E. coli, other enterobacteria, species of Klebsiella, Proteus and Enterobacter, Pseudomonas aeruginosa, Neisseria meningitides and Bacteroides, but also gram-positive bacteria like Staphylococcus aureus, Streptococcus pneumoniae and other streptococci; rare fungi, viruses, or parasites. When the cells of the non-specific immune defense come into contact with lipopolysaccharides (LPS), i.e., components of the bacterial cell wall, peptidoglycans or lipoteichoic acids (LTA), the innate immunity is activated, and in the early phase of the infection cytokines are secreted from various immune cells. Even though these cytokines play an important role in the defensive reaction, activated neutrophils for example are attracted to the site of infection, and the entrance of these cytokines and bacterial substances into the circulatory system produces a chain of unfavorable pathophysiological events, which can lead to death via sepsis and septic shock.
The spectrum of the pyrogenic substances that occur depends on the pathogen or pathogen spectrum. Certain pathogens form pathogen-specific molecular structures or pyrogen patterns, so-called pathogen-associated microbial patterns or PAMPs, which are detected by special, so-called pattern recognition receptors (PRRs) such as toll-like receptors (TLR), NOD-like receptors (NLR), RIG-I-like receptors (RLR), C-type lectin receptors (CLR), cytosolic dsDNA sensors (CDSS), scavenger receptors, mannose-binding lectin 2 (MBL-2) receptor and glucan receptors. Among PRRs, the toll-like receptors (TLR) constitute the largest and best known family.
TLRs are highly conserved transmembrane proteins with leucine-rich extracellular domains and a cytoplasmic domain of approximately 200 amino acids. Because of their homology in the cytoplasmic domain, they belong to the interleukin-1 receptor/toll-like receptor super family. The extra cellular domain is directly involved in detecting different pathogenic molecular structures and therefore constitutes the binding domain of the receptor. TLRs are activated via the binding of PAMPs, i.e., foreign structures. As of today, ten different human TLRs have been identified. They are expressed in different cells types in the immune system, predominantly in monocytes, macrophages, dendritic cells as well as B-cells and T-cells.
TLR 2 is essential for identifying a plurality of PAMPs of gram-positive bacteria, including bacterial lipoproteins and lipoteichoic acids. TLR 3 is involved in identifying double-stranded virus RNA. TLR 4 is activated predominantly by LPS gram-negative bacteria. TLR 5 detects bacterial flagellin. TLR 7 and TLR 8 detect synthetic small antiviral molecules and single-strand RNA. TLR 9 was detected in endoplasmic reticulum (ER) and after stimulation with DNA containing CpG motives, for example CpG oligodeoxynucleotides, is recruited to endosomal/lysosomal compartments. CpG motives are areas within a nucleic-acid strand, in which the building blocks of cytosine (C) and guanine (G) unexpectedly frequently occur (“p” stands for a phosphate group, which connects the two building blocks “C” and “G”); such CpG motives are found especially frequently in the genome of bacteria and viruses, but not those of vertebrates. The specificity of TLRs is expanded by the interaction of two TLRs so that e.g., TLR 2 and TLR 1 are in a position as a heterodimeric molecule to identify triacylated lipoprotein. Dimers of TLR 2 and TLR 6 can identify diacylated lipoprotein.
With the presence of a septic illness or even already with the suspicion of such an illness, treatment must occur at an early stage. Until now, blood cultures from the patient have been used for microbiological testing for diagnosis when there is an indication of sepsis. This process costs valuable time and frequently does not identify the pathogens, because this is possible only with vital pathogens. Pyrogenic substances of the pathogens such as cell wall components cannot be determined with this method.
Currently four commercial detection systems for pyrogens have been approved in corresponding EU and FDA regulations for pyrogens: the rabbit pyrogen test (KPT), limulus amoebocyte lysate test (LAL), the immune pyrogen test (IPT) and the monocyte activation test (MAT).
The rabbit pyrogen test is based on the “fever reaction” of animals to pyrogens. It is an animal test, in which the test substance is applied to the rabbit's ear vein. In order to detect a defensive reaction of the animal's body to the substance, a measurement is taken of the rectal fever after several hours. This test is time-consuming and expensive and associated with the calculated suffering of animals. A further serious disadvantage of this test is the high variance in the results, caused by living and therefore individual organisms. Pyrogens can be detected with it, but they cannot be identified. A test for viruses is also not possible. In addition, transferability to humans is limited, because not all human pyrogenic substances also trigger a fever in a rabbit.
Another known test is the limulus amoebocyte lysate test (LAL, e.g., PyroGene from Lonza; ToxinSensor from GenScript Inc.). This test uses the fact that the haemolymph of the Limulus polyphemus (horseshoe crab) coagulates in the presence of LPS gram negative bacteria. This method is more sensitive and can be standardized better than the known rabbit test, but it detects only LPS gram-negative organisms (limit of detection: 3 pg/mL); other pyrogens remain undetected.
Another known test is the immune pyrogen test, e.g., Endosafe-IPT (Charles River). It is based on the “fever reaction” of human cells to the presence of pyrogens. It is a human whole blood test, in which cytokine IL-1 is secreted from vital blood cells as a response to a pyrogenic substance, which can be determined quantitatively using ELISA (limit of detection: 20-50 pg/mL). This system also detects pyrogens of gram-positive pathogens. However, the test is associated with still a greater amount of time and effort. Human whole blood must be made available, which is a potential human pathogen.
The monocyte activation test (MAT, e.g., PyroDetect System, EMD Millipore Corporation; PyroDetect Kit Biotest AG) is also based on the detection of cytokine IL-1 formed by monocytes using ELISA, wherein in comparison to IPT cryo-blood is used. It is not possible to specify the pyrogens.
In addition to the pharmacopoeia-approved tests that have been described so far, there are other in vitro methods for detecting pyrogens for use in research. These are mammalian cells, which stably express the TLRs and are able to specifically indicate the presence or absence of pyrogens with the aid of a reporter gene (e.g., HEK-blue and RAW-blue TLR cells from InvivoGen). In addition, DE 10 2006 031 483 discloses a cellular pyrogen test, wherein a transgenic NIH-3T3 cell expresses at least one TLR and a reporter gene, which is under the expression control of an NF-κB inducible promoter.
The known tests are time-consuming and require a well-equipped laboratory (ELISA test, human blood processing, animal experiments) as well as expertise in handling cell cultures. For this reason, these test systems are only suited for well-trained users, for example in research or specialized laboratories. As a result, there is a need for a test system for detecting pyrogens that can be conducted quickly and simply. Moreover, there is a need for a test system for specifying pyrogen patterns (PAMPs) in order to be able to draw conclusions from them about the pathogens or pathogen spectrum.
Furthermore, medical technology products and pharmaceuticals must be tested for the absence of pyrogens in order to prevent pyrogens from getting into the bloodstream in this manner. The absence of pyrogens is therefore a mandatory prerequisite for the use of these types of products on the body. There is a need for improved tests for pyrogenic residues on medical instruments, donor tissue, injectable drugs and medical products such as implants or instruments (catheters, etc.) There is also a need in the food industry and pharmaceutical industry for the improved detection of pyrogenic substances and microbes and the identification thereof in foodstuffs, food ingredients, raw materials, and starting materials for foodstuffs or drugs.